ABSTRACT Ambient ozone is associated with increased respiratory illness, hospitalizations, and cardiovascular mortality. Because the lung is commonly exposed to low level inhaled ozone, understanding how ozone modifies innate immune mechanisms is of considerable importance to human health. Previously, we have identified that ozone enhances pulmonary innate immune response to lipopolysaccharide (LPS) including; cellular inflammation, cytokine production, lung injury, cellular apoptosis, and airway hyper-responsiveness. We discovered that ozone-priming of innate pulmonary immunity, in part, involved enhanced surface expression of tlr4 on alveolar macrophages, which amplifies the biological response to inhaled LPS. Oxidant-dependent release of extracellular matrix hyaluronan fragments induces trafficking of tlr4 to the cell surface in a manner dependent on activation of NF-kB. However, the intracellular signaling pathways required for ozone-induced alterations in tlr4-signaling remain unknown. We now identify that inhalation of ozone results in robust increased levels of NAD+ and NADP+ in alveolar macrophages. The focus of this competitive renewal is understanding the role of specific NAD(P)H-dependent enzymes in ozone-induced alterations in innate immunity. Mechanistic understanding of the pathways that regulate macrophage-derived toll-like receptor 4 signaling after exposure to this common air pollutant is of considerable interest in the pathogenesis of airways disease. Our overall hypothesis is that macrophage-derived innate immune response after inhalation of ambient ozone is dependent on NAD(P)H-dependent enzymes including both PKC-dependent activation of NOX2 resulting in enhanced intracellular ROS and NQO1-dependent alterations in global chromatin structure in both mice and humans. We will address this hypothesis through the following Specific Aims. Specific Aim 1. Determine the role of NOX2 (gp91phox/p47phox) in the biological response to inhaled ozone and tlr4-dependent signaling. Specific Aim 2. Determine the role of NQO1 in ozone regulation of innate immunity through modifications in SIRT1-dependent alterations in chromatin structure. Specific Aim 3. Determine whether inhaled ozone modifies NAD(P)+/NAD(P)H and the function of NOX2 and NQO1 in primary alveolar macrophages from healthy human subjects.